This application claims priority from Korean Patent Application No. 2003-56432, filed on Aug. 14, 2003, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
1. Field of the Invention
The present invention relates to a primer set selected from the group consisting of primers having nucleotide sequences as set forth in SEQ ID NOS: 1 through 40, a method for detecting hepatitis B virus in which PCR is performed using the primer set, and a hepatitis B virus detection kit including the primer set.
2. Description of the Related Art
Hepatitis B is a representative virus infectious disease prevalent worldwide. It is one of common infectious diseases, ranking ninth as a cause of death. More than 300 millions of the global population is chronic hepatitis B virus carriers. Therefore, chronic hepatitis, hepatocirrhosis, liver cancer, and the like cause a million of death per a year. In this country, it is known that about 5-10% of the entire population is hepatitis B virus carriers.
Hepatitis B virus (HBV) is a virus that is consisted of viral proteins including three antigen proteins, HBsAg, HBcAg, and HBeAg. HBV DNA is protected by a protein structure called as core antigen (HBcAg) and a core is enveloped with an envelope protein known as surface antigen or S antigen (HBsAg).
As detection methods for hepatitis B, there have been widely used liver function tests that quantify T protein, albumin, T bilirubin, sGOT, sGPT, r-GPT, or alkaline phosphatase (ALP), serological detection of HBsAg and HBeAg using enzyme immunoassay, and the like. However, these methods are effective in detecting advanced hepatitis B but have problems such as difficulty of early diagnosis and low reliability.
Therefore, recently, there have been widely used a hybrid capture assay, a branched DNA assay, and a genetic assay based on polymerase chain reaction (PCR). In particular, the PCR-based genetic assay is preferred because it has high sensitivity and rapidity, and a pharmacological action against hepatitis B can be understood by observing the degree of proliferation of HBV.
PCR is a technique in which when a primer set binds to complementary strands of target nucleic acid sequence, a gene between the two primer binding sites is amplified to more than several hundreds of thousands of copies within a short time. PCR is well known in the pertinent art. In PCR, two nucleic acid primers complementary to corresponding strands of a target nucleic acid sequence are annealed to denatured strands of the target nucleic acid sequence under hybridization conditions, and then DNA polymerase, which is normally thermal stable, initiates extension at the ends of the hybridized primers to obtain DNA double strands. The above procedure is repeated to multiply the target nucleic acid sequence. When the nucleic acid primers are not hybridized with the target nucleic acid sequence, no amplified PCR products are obtained. In this case, the PCR primers serve as hybridization probes.
The PCR products thus amplified can be detected by observing labeled nucleotides in amplified strands using labeled primers. The labeled primers may be primers labeled with radioactive substance, fluorescent dye, digoxygenin, horseradish peroxidase, alkaline phosphatase, acridium ester, biotin, and jack bean urease, but are not limited thereto. PCR products obtained using non-labeled primers can be detected by visualization with dye after gel electrophoresis.
However, with respect to diagnosis of hepatitis B based on these PCR techniques, there may arise problems in that a long gene amplification time of 2-3 hours is required, false positive or false negative results may be yielded, and accurate diagnosis is difficult due to the presence of different genotypes. Therefore, to increase the diagnostic accuracy for hepatitis B, detection methods using both PCR and hybridization have been suggested. However, these detection methods also require an excessively long detection time and are costly.